CN116458424B - Tissue culture and rapid propagation method for small-fruit sweet persimmon - Google Patents
Tissue culture and rapid propagation method for small-fruit sweet persimmon Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a tissue culture and rapid propagation method of small-fruit sweet persimmon, which comprises the following steps: primary culture: taking the stem with buds, sterilizing, stripping a plurality of layers of scales outside the full dormant buds, inoculating the stem with buds into a primary culture medium, and culturing to obtain primary tissue culture seedlings; proliferation culture: cutting the primary tissue culture seedling into stem segments, inoculating the stem segments into a proliferation culture medium containing betaine for culture to obtain a root-free tissue culture seedling; rooting culture: rooting culture is carried out on the rooting-free tissue culture seedlings by adopting a dipping rooting method or a two-step rooting method, so that rooting tissue culture seedlings are obtained; wherein, the first plant growth regulator adopted in the dipping rooting method is NAA or IBA; the second plant growth regulator adopted in the two-step rooting method is a mixture of NAA, IBA and MT; domestication and transplanting: taking rooting tissue culture seedlings, closing bottles, hardening the seedlings, opening the cover, hardening the seedlings, transplanting the seedlings to seedling trays for culturing until the seedlings survive, and transplanting the seedlings to a field. The invention can effectively shorten the time of finished persimmon stock seedlings and solve the problem of difficult rooting in the traditional persimmon in-vitro regeneration process.
Description
Technical Field
The invention belongs to the field of plant cultivation, and particularly relates to a tissue culture and rapid propagation method of small-fruit sweet persimmon.
Background
The cultivated persimmon is mainly bred through grafting, and can be crisp and eaten after the persimmon is completely ripe. At present, the persimmon stocks in China mainly comprise Jun-qian, oil persimmon, wild persimmon, american persimmon and Zhejiang persimmon (Luo Zhengrong and Wang Renzi, 2001). The most widely used rootstock monarch and the current main cultivated persimmon high-quality variety 'rich' system grafting are incompatible, and become important factors for limiting the development of the persimmon.
In recent years, research shows that the small-fruit sweet persimmon has strong grafting affinity with the main-pushing rich variety, and has important value in the cultivation and popularization of the sweet persimmon. The agricultural university persimmon research team in China collects the novel genotypes of complete sweet persimmon broad-affinity stock small fruit sweet persimmon, cow-eye persimmon and the like from Dabie mountain area in Hubei province, and finds that the small fruit sweet persimmon has grafting affinity with 'secondary Lang', 'taiqiu', 'rich' series sweet persimmon (Hu Meng, chen Li, liu Yifeng, zhang Qinglin, luo Zhengrong.2017) 'small fruit sweet persimmon' and 'cow-eye persimmon' as application potential researches of complete sweet persimmon stocks through field performance investigation of isolated grafting affinity identification, callus grafting experiments and 6-year-old stock spike complex. At present, small-fruit sweet persimmon is used as a broad-affinity stock, the propagation mode is mainly seed sowing, the time required for the finished product of persimmon stock seedlings bred by the conventional method is long, the offspring are easy to generate variation and character separation due to chromosome recombination, the consistency of the seedlings is low, and the management is complicated; persimmon in vitro culture can solve the problem.
However, the problem of difficult rooting of persimmon is generally considered in the tissue culture process, and the problem is a great obstacle for commercial production of persimmon seedlings. The factors causing the difference of rooting rate of persimmon tissue culture seedlings include various factors such as variety type, culture medium type, hormone type, treatment time and treatment method, tissue culture Miao Jidai times and the like; wherein the tissue culture of persimmon is variety-different. TAO and the like test the influence of variety genotypes on adventitious bud formation by using calli derived from 16 varieties as test materials, and find that the appearance conditions such as colors and the like of calli derived from different varieties are greatly different, and only 8 varieties can form adventitious buds, and the formation percentage of the adventitious buds is different from 2% to 72%, and presumably the reason is that the proliferation capacity of different varieties is different, or that the same culture medium is used in the test, but the requirements of different varieties on the culture medium are different. In addition MURAYAMA sweet persimmon varieties are subjected to rooting culture by adopting the same method, the rooting rate is also greatly different from 8-96%, and the existence of variety difference in the persimmon tissue culture process is again proved. At present, a high-efficiency and large-scale small-fruit sweet persimmon stock propagation technology system is lacking, and no report on in-vitro regeneration of an intact individual of the small-fruit sweet persimmon is available.
Disclosure of Invention
The invention aims to overcome the technical defects, and provides a tissue culture and rapid propagation method for small-fruit sweet persimmon, which solves the technical problems of long time, multiple variation and difficult rooting when in-vitro culture is adopted for the finished product of persimmon stock seedlings caused by propagation of small-fruit sweet persimmon seeds in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention provides a tissue culture and rapid propagation method for small-fruit sweet persimmon:
The method comprises the following steps:
(1) Primary culture: taking the stem with buds, sterilizing, stripping a plurality of layers of scales outside the full dormant buds, inoculating the stem with buds into a primary culture medium, and culturing to obtain primary tissue culture seedlings;
(2) Proliferation culture: cutting the primary tissue culture seedling into stem segments, inoculating the stem segments into a proliferation culture medium containing betaine for culture to obtain a root-free tissue culture seedling;
(3) Rooting culture: rooting culture is carried out on the rooting-free tissue culture seedlings by adopting a dipping rooting method or a two-step rooting method, so that rooting tissue culture seedlings are obtained; wherein, the first plant growth regulator adopted in the dipping rooting method is NAA or IBA; the second plant growth regulator adopted in the two-step rooting method is a mixture of NAA, IBA and MT;
(4) Domestication and transplanting: taking rooting tissue culture seedlings, closing bottles, hardening the seedlings, opening the cover, hardening the seedlings, transplanting the seedlings to seedling trays for culturing until the seedlings survive, and transplanting the seedlings to a field.
Further, in the step (1), the bud-bearing stem section is obtained by collecting strong and full-bud branches in winter; the sterilization is to dip and wash the bud stem with Tween-20 for 1-2 min and then clean with sterile water; the disinfection is to soak the sterilized bud-bearing stem in 75% alcohol solution for 25-35 s, disinfect in 0.5-1.2% sodium hypochlorite solution for 5-7 min and then clean with sterile water.
Further, in the step (1), 2-3 layers of scales outside the full dormant buds are stripped and inoculated in a primary culture medium; the primary culture medium consists of MS (1/2N) +1mg/L ZT+0.1mg/L IAA+30mg/L sucrose+7 g/L agar+0.6 g/L PVP-40; the culture conditions of the primary culture are that the primary culture is carried out in a tissue culture room with the temperature of 25+/-2 ℃ and the illumination intensity of 1000-3000 lx and the illumination time of 16h/d, and the culture time is 30-60 d.
Further, in the step (2), the primary tissue culture seedling is sheared into stem segments with the length of 1 cm to 1.5 cm.
Further, in the step (2), the proliferation medium containing betaine consists of DKW+1mg/L ZT+0.1mg/L IAA+30mg/L sucrose+7 g/L agar+0.6 g/L PVP-40+100-1000 mu mol/L betaine; the culture conditions of the proliferation culture are the same as those of the primary culture, and the culture time is 30-60 d.
Further, in the step (3), the dipping rooting method is to firstly soak the basal part of the rooting-free tissue culture seedling with a first plant growth regulator, then carry out dark treatment for 5-15 d, and then transfer the basal part into a basic culture medium A for rooting culture for 30-50 d; when the first plant growth regulator is NAA, the concentration is 50-200 mg/L, and the soaking time is 5-15 min; when the first plant growth regulator is IBA, the concentration is 100-200 mg/L, and the soaking time is 5-10 min. The basic culture medium A consists of 1/2MS+1mg/L active carbon+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40.
Further, in the step (3), the two-step rooting method is to inoculate the radicle tissue culture seedling into the basic culture medium B, carry out the dark treatment for 5-15 d, and then transfer the radicle tissue culture seedling into the basic culture medium A for rooting culture for 30-50 d; the basic culture medium B consists of basic culture medium A+NAA+0.2-1.5 mg/L and IBA+0.1-5 mg/L MT.
Further, in the step (4), the seedling hardening time of the closed bottle is 3-10 d, and the seedling hardening time of the open cover is 2-4 d.
Further, in the step (4), the culture medium transplanted to the seedling culture hole tray adopts mixed soil of the seedling culture medium and vermiculite, and the mixed soil is mixed with 1000 times carbendazim before cultivation; after transplanting to seedling tray, covering with air-permeable heat-insulating cover, adding water into tray; spraying 1000 times carbendazim solution every 7d, and culturing under natural illumination at 23-25 ℃ until survival.
Compared with the prior art, the invention has the beneficial effects that:
The invention obtains the radicle-free tissue culture seedling through primary culture and multiplication culture, and improves the multiplication efficiency of the small fruit sweet persimmon by adding betaine in the multiplication culture; the rooting method by dipping the first plant growth regulator or the two-step rooting method by the second plant growth regulator can induce rooting, promote plant roots to be thick and strong, and is beneficial to improving survival rate; finally, by adopting the cooperation of closed bottle seedling hardening and open cover seedling hardening, the tissue culture seedling of the small fruit sweet persimmon is successfully domesticated, and a complete persimmon in-vitro culture propagation system is constructed; compared with the propagation mode of seed sowing, the method can effectively shorten the finished product time of persimmon stock seedlings, solves the problem that the traditional persimmon in-vitro regeneration is difficult to root, has high consistency of the obtained offspring seedlings, establishes a high-efficiency 'small fruit sweet persimmon' in-vitro rapid propagation technology system, and provides technical support for sustainable development of the high-quality sweet persimmon industry in China.
Drawings
FIG. 1 is a schematic diagram showing the transplanting of the small-fruit sweet persimmon in example 1 of the present invention to a field.
FIG. 2 shows the growth state of the persimmon explants treated with sodium hypochlorite at different concentrations; wherein A: treating the small fruit persimmon explant with 0.5% sodium hypochlorite; b: treating the small fruit sweet persimmon explant with 1% sodium hypochlorite; c:2% sodium hypochlorite treatment of the small fruit sweet persimmon explant; d:2.5% sodium hypochlorite treatment of the small fruit sweet persimmon explant.
FIG. 3 shows the growth of tissue culture seedlings after 30d treatment with betaine at different concentrations; wherein, A is that 0 mu mol/L betaine is used for treating tissue culture seedlings; b, treating tissue culture seedlings by 10 mu mol/L betaine; c, treating the tissue culture seedlings by 50 mu mol/L betaine; d, treating the tissue culture seedlings by using 100 mu mol/L betaine; e, 1000 mu mol/L betaine treatment of the tissue culture seedlings; f, 2000 mu mol/L betaine treatment of the tissue culture seedlings.
FIG. 4 shows rooting states of the small fruit sweet persimmon after different treatments (dipping method); wherein a 1-a9 is dipping treatment 1-9.
FIG. 5 shows rooting states of the sweet persimmon subjected to different treatments (two-step rooting method); wherein b 1-b9 is a two-step rooting method treatment 1-9.
FIG. 6 is a schematic diagram of the rapid propagation system of Diospyros kaki of the present invention wherein, a. Dormancy buds; b, sterilizing with 75% alcohol; c, sterilizing with 1% sodium hypochlorite; d. the surgical knife strips a plurality of layers of scales outside the dormant bud; e. inoculating into a culture medium; f. adventitious bud formation and elongation (30 d culture); g. primary culturing stem segments (1-1.5 cm) with 4-5 leaves; h. primary culturing stem segments with buds; i. performing primary culture for 30d, and then performing tissue culture seedling; j. subculturing for 30 days and then carrying out bud stem segment; k. the stem with 4-5 leaves (2-3 cm long) after 30d of subculture; i, rooting tissue culture seedlings; m, domesticating tissue culture seedlings; n. transplanting the plants after 2 months.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The technical scheme of the invention is conventional in the field unless specifically stated otherwise, and the reagents or materials are commercially available unless specifically stated otherwise.
Interpretation of terms and abbreviations
MS: MS culture medium;
MS (1/2N): MS culture medium, nitrogen element halving.
1/2MS: the macroelements in MS culture medium are halved.
ZT: zeatin;
IAA: indoleacetic acid as an auxin;
PVP: polyvinylpyrrolidone;
DKW: DKW medium;
NAA: naphthalene acetic acid;
IBA: indolebutyric acid;
MT: n-acetyl-5 methoxy tryptamine, also known as melatonin.
Basal callus induction rate calculation mode: callus induction = (number of explants forming callus/total number of explants) ×100%.
The method for calculating the regeneration rate of the dormant buds comprises the following steps: regeneration = (number of explants germinated shoots/number of total explants) ×100%.
Pollution rate = number of polluted buds/total buds.
Mortality = number of sterilized dead shoots/total shoots.
Survival = number of surviving shoots per total shoots.
The test stock variety of the invention is Diospyros kaki (Diospyros kaki th.) Thunb. Xiaoguo Tianshi'), the explants are the movable buds of adult Diospyros kaki, and the explants are taken from the germplasm resource nursery of agricultural university in Wuhan Hua in Hubei province. Selecting a tissue culture seedling which grows robustly, and taking young shoots with the length of 2.0-3.0cm as a rooting material.
The invention discloses a tissue culture and rapid propagation method of small-fruit sweet persimmon, which comprises the following steps:
(1) Primary culture
And collecting strong and full shoots in a winter bud dormancy period, cutting the shoots into stem segments with buds, placing the stem segments into a sterilized conical flask, immersing and washing the conical flask for 1-2 min by using Tween-20, and washing the conical flask for 2-3 times by using sterile water. Before inoculation, soaking in 75% alcohol solution for 25-35 s, preferably 30s, sterilizing in sodium hypochlorite with concentration of 0.8-1.2% (preferably 1%) for 5-7 min (preferably 6 min), washing with sterile water for several times until the water in the conical flask is colorless, stripping off the outer layers of scales of full dormant buds by a surgical knife, inoculating in MS (1/2N) +1mg/L ZT+0.1mg/L IAA+30mg/L sucrose+7 g/L agar+PVP-40 (0.6 g/L) culture medium, and culturing in a tissue culture room with illumination intensity of 2000lx and 16h/d at the temperature of 25+/-2 ℃. And inoculating 1 explant to each bottle, inoculating 20 bottles after each treatment, repeating for 3 times, and culturing for 30 days to obtain the explant of the primary tissue culture seedling, wherein the pollution rate and the death rate are counted.
(2) Proliferation culture
Cutting the primary tissue culture seedling obtained by primary culture into stem segments of 1-1.5cm, inoculating the stem segments into a proliferation culture medium which is formed by adding betaine (preferably 100-1000 mu mol/L) with different concentrations into DKW+1mg/L ZT+0.1mg/L IAA+30mg/L sucrose+7g/L agar+PVP-40 (0.6 g/L) for combination, and culturing under the same culture conditions as the primary culture. 1 stem segment is inoculated in each bottle, 20 bottles are inoculated in each treatment, 3 times are repeated, and the explant of the root-free tissue culture seedling is obtained after 30-60 days of culture, and the tillering number, the leaf number, the stem node number and the plant height of the explant of each root-free tissue culture seedling are counted.
(3) Rooting culture
The invention utilizes two methods for rooting culture, which concretely comprises the following steps:
Dipping rooting method: dipping the root-free tissue culture seedling explants (1-3 cm in length) with plant growth regulators of different types and concentrations on the root parts of the young shoots by using 1/2MS+1mg/L active carbon+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40 as a basic culture medium A, carrying out dark treatment for a certain period of time, and then transferring the young shoots into the basic culture medium A without the plant growth regulator to promote rooting of the young shoots; preferably, the plant growth regulator comprises growth hormone such as NAA or IBA; the concentration is 50mg/L to 200mg/L; further, the NAA concentration is preferably 50-200 mg/L, and the treatment time is preferably 5-15 min; the concentration of IBA is preferably 100-200 mg/L, and the treatment time is preferably 5-10 min.
Two-step rooting method: adding plant growth regulators of different types and different concentrations into a basic culture medium A (basic culture medium of the same dipping rooting method) to obtain a basic culture medium B, inoculating the root-free tissue culture Miao Qiequ callus with the length of 1-3 cm obtained in multiplication culture into the basic culture medium B, performing dark treatment for different days, and transferring into the basic culture medium without the plant growth regulators to induce rooting; preferably, the plant growth regulator comprises a mixture of growth hormone such as NAA, IBA and MT, the concentration of the mixture is respectively 0.1mg/L to 5mg/L, and the minimal medium B is preferably NAA+0.5 to 1mg/L and IBA+0.2 to 1.5mg/L and MT; the number of days for dark treatment is 5-15 days.
In the rooting culture, the dark treatment is shading, the temperature is 23-25 ℃, and the humidity in a tissue culture bottle is constant at 80-85%.
Two induction rooting methods are to inoculate 5 stem segments in each bottle, inoculate 6 bottles in each treatment, repeat for 3 times, count rooting rate 30d after inoculation, and measure root length, surface area, volume, branch number and root tip number of root tissue culture seedling root system by using Epson Scan root system scanner (support software is WinRHIZO).
(4) Transplanting test
The rooting tissue culture seedling is domesticated and transplanted, the rooting tissue culture seedling is placed in a growth chamber, closed bottle seedling hardening is carried out for 7d, then cover is opened for hardening for 3d, the culture medium is mixed with mixed soil of a commodity substrate and vermiculite (10:1), 1000 times carbendazim is used for mixing soil before cultivation, a transplanting container opening is 6cm, a plastic seedling raising plug tray with the height of 11cm is provided with a plastic cover with the depth of 10cm, a certain amount of water (a certain humidity is kept by evaporation, preferably the humidity is 80% -85%) is added into the tray after transplanting, 1000 times carbendazim solution is sprayed every 7d, and water is properly supplemented into the tray. Culturing at 23-25 deg.c under natural light to survive, observing and recording the growth state of seedling, and counting the survival rate and plant height of seedling after 60 d.
(5) Data processing
And summarizing the obtained data. And counting the rooting rate, the rooting number and the longest root length of each treatment, and carrying out variance and range analysis on the data by adopting SPSS STATISTICS statistical software. And carrying out arcsine conversion on the rooting rate before analysis of variance, and carrying out multiple comparison by adopting Dancan method. And simultaneously, comprehensively evaluating the rooting effect by a membership function method. Membership function value calculation formula: r (X i)＝(Xi-Xmin)/(Xmax-Xmin), i=1, 2,3, …, n.
Wherein Xi is an index measurement value; x min and X max are the minimum and maximum values, respectively, of a certain index in different treatments. The measured value X i is converted into a corresponding membership function value R (X i) by using a formula, and the comprehensive membership function values of the indexes of different treatments are summed and calculated.
The present invention will be described in further detail with reference to specific examples.
Example 1:
The tissue culture and rapid propagation method of the small-fruit sweet persimmon comprises the following steps:
A tissue culture regeneration rapid propagation method for small-fruit sweet persimmon comprises the following steps:
1) Primary culture: collecting strong and full-bud branches at the early growth stage, cutting into stem segments with buds, placing into sterilized conical flasks, soaking and washing for 1-2 min with Tween-20, and washing with sterile water for 2-3 times. Before inoculation, soaking in 75% alcohol solution for 30s, sterilizing in 1% sodium hypochlorite for 6 min, washing with sterile water for several times until the water in the conical flask is colorless, stripping 2-3 layers of scales outside full dormant buds with a surgical knife, inoculating in MS (1/2N) +1mg/L ZT+0.1mg/L IAA+30mg/L sucrose+7 g/L agar+0.6 g/L PVP-40 culture medium, adjusting pH to 5.8-6.0, and culturing in a tissue culture chamber at 25+ -2deg.C and illumination intensity 2000lx and 16 h/d. 1 explant is inoculated to each bottle, 20 bottles are inoculated to each treatment, the culture is repeated for 3 times, after 30d of culture, the induction rate of the callus at the basal part of the tissue culture seedling is counted to be 100%, the pollution rate is 23.6%, the death rate is 13.84%, and the survival rate is 62.56%.
2) Proliferation culture: cutting the primary tissue culture seedling obtained by primary culture into stem segments with the length of 1-1.5 cm, inoculating the stem segments into a tissue culture chamber with the temperature (25+/-2) DEG C, the illumination intensity of 2000lx and 16 hours/d, wherein the stem segments are cultured in a DKW+1mg/L ZT+0.1mg/L IAA+30mg/L sucrose+7 g/L agar+0.6 g/LPVP-40+1000 mu mol/L betaine proliferation medium. 1 stem segment was inoculated per flask, 20 flasks were inoculated per treatment, 3 replicates, and cultured for 30d. And (3) carrying out secondary culture propagation on part of the aseptic seedlings with a period of 30d, namely cutting the aseptic seedlings into stem segments (containing 2-3 axillary buds) with a length of 1-2 cm, placing the stem segments in a secondary culture medium (the same as betaine proliferation culture medium) for secondary culture, wherein the induction rate of callus at the base of the tissue culture seedlings is 100%, the regeneration rate of cluster buds is 100%, the average proliferation coefficient is 3.7, the plant height is 3.29cm, and the number of leaves is 9.49 pieces/plant.
The other part of aseptic seedlings is directly inoculated to a rooting medium after being cultured for 30d in a secondary culture medium.
The DKW culture medium is utilized to proliferate the tissue culture seedlings, so that the propagation coefficient of the tissue culture seedlings of the small-fruit sweet persimmon is improved, and the robust tissue culture seedlings of the small-fruit sweet persimmon are cultivated.
3) Rooting culture
Rooting culture is carried out by utilizing a dipping rooting method (culture 30 d): and (3) treating the sterile seedlings cultured for 30d by a dipping rooting method, inoculating the treated sterile seedlings into a rooting culture medium for rooting culture, inoculating 5 stem segments in each bottle in rooting culture, inoculating 6 bottles in each treatment, repeating for 3 times, counting the rooting rate for 30d after inoculation, and measuring the length, the surface area and the volume of root systems of the rooting tissue culture seedlings by adopting an Epson Scan root system scanner (supporting software is WinRHIZO).
Specific conditions of the dipping rooting method: 1/2MS+1mg/L active carbon+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40 is used as a basic culture medium, 200mg/L NAA is used for soaking the basal part of the young shoot for 15min, dark treatment is carried out for 10d, and then the young shoot is transferred into the basic culture medium without plant growth regulator to promote rooting.
Through tests, the average total length of the most robust root system reaches 18.30cm, the average surface area of the root system is 7.70cm 2, and the average volume of the root system is 0.20cm 3.
4) The rooting tissue culture seedling obtained by dipping rooting method is domesticated and transplanted, the tissue culture seedling is put in a growth chamber, closed bottle seedling hardening is carried out for 7d, then cover hardening is carried out for 3d, the culture medium is selected from mixed soil (mass ratio of 10:1) of commercial road medium (source is Shandong commercial road biotechnology company) and vermiculite, soil mixing is carried out by 1000 times of carbendazim before cultivation, a transplanting container mouth is 6cm, the height is 11cm, a plastic seedling raising cave dish with the depth of 10cm is matched, the plastic cover with a ventilation heat preservation cover is used after transplanting, a certain amount of water is added into the tray (certain humidity is kept by evaporation), 1000 times of carbendazim solution is sprayed every 7d, and water is properly added into the tray. Culturing under natural light condition of 25deg.C until the seedlings survive, observing and recording the growth condition of the seedlings, and keeping the survival rate of the seedlings at 52.5% after 60d, wherein the seedlings grow well and can grow normally after transplanting to field.
Compared with the prior art which is mostly in laboratory environment and fails to be domesticated and transplanted to the field, the success rate of the seedling hardening from domestication and hardening to transplanting to the field reaches 52.5 percent, and as shown in figure 1, the seedlings of the small fruit sweet persimmon can be completely suitable for field planting; after transplanting in a field for 8 months, the average plant height of the small fruit persimmon is up to 49.17cm, the stem thickness is up to 6.01mm, and the method can be completely used for grafting the persimmon.
Example 2 (investigating the Effect of sodium hypochlorite concentration on explant sterilization and Primary culture)
Example 2 differs from example 1, step 1) only in that: sodium hypochlorite concentrations were adjusted to 0.5%, 2% and 2.5%, respectively, with the other conditions being the same as in example 1, step 1). The results of the test for the effect of treating the sweet persimmon explant are shown in the following table 1 and fig. 2.
TABLE 1 Effect of sodium hypochlorite at different concentrations on the treatment Effect of Diospyros kaki explants
Note that: each of the contamination rate, mortality, survival rate was calculated from the average of three raw data.
The surface sterilization of the explant is the key to establishing a micro-propagation system of the small-fruit sweet persimmon, and the subsequent growth of the persimmon tissue culture seedling can be greatly influenced by the existence of germs, so that the acquisition of the small-fruit sweet persimmon sterile material can be prevented. The experimental results show that the disinfection time is the same, the pollution rate is reduced from 26.11% to 0 as the concentration of sodium hypochlorite is increased, but the tissue damage to the explant is increased as the concentration of sodium hypochlorite is increased, most of pollutants are removed in the process of disinfecting the explant by using 2% sodium hypochlorite solution, and meanwhile, larger damage is caused to the explant, and the explant differentiates some calluses after 7d, as shown in fig. 2C; the explant can be effectively removed pollutants by soaking in 2.5% sodium hypochlorite solution, but at the same time, plant tissues are completely damaged, the explant turns black and dies more than 1 week after induction, as shown in fig. 2D; the explant sterilized by 0.5% sodium hypochlorite has a high pollution rate, non-embryogenic callus appears after 30d after survival, and endophyte pollution is easily caused after survival, as shown in fig. 2A; the survival rate of 1% sodium hypochlorite is highest, embryogenic callus appears and leaves are differentiated after 30d, and as shown in fig. 2B, a small amount of endophyte pollution exists, and the influence of endophytes needs to be eliminated by continuously changing the culture medium. During primary culture, stem tips are stripped and subjected to primary culture in a culture medium of MS (1/2N) +sucrose (30.0 g/L) +agar (7.0 g/L) +ZT (1.0 mg/L) +IAA (0.1 mg/L) +PVP-40 (0.6 g/L), and the surviving explants can grow normally.
Example 3 (investigating the effect of betaine concentration on proliferation during proliferation culture)
Example 3 differs from example 1, step 2) only in that: the betaine concentrations were adjusted to 0, 10, 50, 100 and 2000. Mu. Mol/L, respectively, and the other conditions and procedures were the same as in example 1. The results of the test for proliferation of the tissue culture seedlings are shown in table 2 and fig. 3.
TABLE 2 Effect of betaine treatments at different concentrations on proliferation of Diospyros kaki tissue culture seedlings
In the betaine concentration range of 100 to 1000 mu mol/L, the tillering number, plant height, stem node number, leaf number and concentration are all positively correlated, and as can be seen from Table 2, the betaine with the concentration of 10, 50, 100 and 2000 mu mol/L has no significant difference from the control in tillering number, so that the betaine has no obvious effect on tissue culture seedling proliferation at lower concentration or above a certain concentration, and the proliferation effect is increased and then reduced in the concentration range of 0 to 2000 mu mol/L. The proliferation effect of the treatment at a betaine concentration of 1000. Mu. Mol/L was optimal, the average number of tillers to be proliferated was 1.59, the plant height was 32.87mm, the number of leaves was 9.49 pieces/plant, and the number of stem nodes was 7.40/plant (Table 4). The analysis of variance results showed that the plant heights of the tissue culture seedlings of the sweet persimmon were not significantly different under the treatment of betaine at different concentrations, and the betaine at 1000. Mu. Mol/L was not significantly different from the treatment at 100. Mu. Mol/L, but the tiller number, leaf number and stem node number were significantly different from those of the other treatments (Table 4). As shown in figures 3A to 3F, after betaine treatment for 30D at different concentrations, the plant growth condition is good, the leaf color is dark green, which shows that the components contained in the basic culture medium DKW can meet the requirement of the growth of the small fruit sweet persimmon, and the proliferation condition and the growth condition of the tissue culture seedlings are obviously superior to those of other groups (the groups shown in figures 3A to 3D and the group shown in figure 3F) after 1000 mu mol/L concentration treatment shown in the group shown in figure 3E, and the group shown in the figure 3D is next.
Example 4 (investigating the effect of IBA, NAA and MT on rooting in the dipping treatment)
Example 4 differs from example 1, step 3) only in that: the plant growth regulator type (NAA, IBA or MT) and the amount were adjusted, and the other conditions and procedures were the same as in example 1. Specific types and amounts are shown in Table 3, and rooting test results are shown in tables 4 to 7 and FIG. 4.
TABLE 3 orthogonal design test factors and level for dip rooting
TABLE 4 rooting properties under different treatments and multiple comparisons (dipping method)
The injection is that A: a hormonal species; b: hormone concentration (mg/L); c, processing time; different letters in the same column represent significant differences (p.ltoreq.0.05).
TABLE 5 analysis of variance (dipping method) of various indexes of rooting of Diospyros kaki Thunb by different treatments
Note that x represents a significant correlation with p <0.05
TABLE 6 various index membership function values of rooting of Diospyros kaki (dipping method)
TABLE 7 comprehensive membership function range analysis (dipping method)
Note that K j is the sum of p values at different levels for the same factor. kj is the average of p values at different levels for the same factor, and R j is very poor.
Analysis of the effect of IBA, NAA and MT dip treatment on rooting:
as shown by the multiple comparative analysis of Table 4 and FIG. 4, the root length effect is preferably 100mg/L NAA for 10min (dipping treatment 2), the main root length reaches 23.17cm, and there is no significant difference from 200mg/L NAA for 15min (dipping treatment 3), but there is significant difference from the other treatment groups.
The analysis of variance results in table 5 shows that the influence of different treatment factors on the root length of the tissue culture seedling is different, the F ratio of each treatment factor is in the order of hormone type, treatment time and regulator mass concentration from large to small, and the hormone type, treatment time and mass concentration obviously influence the root length (p is less than 0.05); the hormone type obviously affects the surface area and the volume of the root system, and the treatment time is obviously affected by the volume of the root system (p is less than 0.05) besides the hormone type. The optimal surface area and volume of the root system is 200mg/L NAA for 15min (dipping treatment 3), the surface area of the root system reaches 7.70cm 2, the volume of the root system is 0.20cm 3, and the surface area and volume of the root system treated by 200mg/L NAA for 15min are not obviously different from those of other treatments in the group, but are obviously different from those of other treatments.
The rooting rate of NAA treatment alone is 82.1% on average, which is obviously higher than that of other two hormone treatments, wherein the rooting rate effect of NAA dipping treatment of 100mg/L for 10min is highest and reaches 90%, variance analysis is shown in table 5, the ratio of each treatment factor F is the maximum mass concentration of the regulator, the mass type of the regulator is the same as the ratio of the treatment time F, and the rooting rate is obviously influenced by the hormone type, the treatment time and the mass concentration of the regulator.
As shown by the analysis, the preferred plant growth regulators in the dipping rooting are NAA and IBA, the concentration of NAA is preferably 50-200 mg/L, and the treatment time is preferably 5-15 min; the concentration of IBA is preferably 100-200 mg/L, and the treatment time is preferably 5-10 min; the rooting rate of the invention reaches 65.56-90.00%, the average total length of the most robust root system reaches 12.01-23.17 cm, the average surface area of the root system is 4.09-7.70 cm 2, and the average volume of the root system is 0.12-0.20 cm 3.
Example 5 (two-step rooting)
Example 5 differs from example 1, step 3) only in that: rooting culture is carried out by adopting a two-step rooting method, and other steps and conditions are the same as in example 1.
Specific conditions of the two-step rooting method: the root-free seedling obtained in the proliferation culture is cut off, the callus is connected to a rooting culture medium of 1/2MS+0.5mg/L NAA+0.2mg/L IBA+5mg/L MT+1mg/L active carbon+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40, and the rooting culture medium is transferred to a basic culture medium of 1/2MS+1mg/L active carbon+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40 after 10 days of dark treatment to induce rooting.
Through tests, the rooting rate reaches 71.50%, the average total length of the most robust root system reaches 24.14cm, the average surface area of the root system is 9.74cm 2, and the average volume of the root system is 0.27cm 3.
Example 5 compared with example 1, the rooting rate by adopting the two-step rooting method is slightly reduced, but the root system is the most robust, and the average total length, the average surface area and the average volume of the root system are obviously improved; compared with the prior art (establishment of a tissue culture and rapid propagation system of Yihe ancient persimmon trees, zheng Anshun and the like), the adventitious root aseptic seedlings prepared by the method are thicker in roots, more in root systems, easier to survive and good in tolerance.
Example 6 (investigating the effect of dark treatment time and plant growth regulator concentration on rooting in a two-step rooting method)
Example 6 differs from example 5, step 3) only in that: the dark treatment time, plant growth regulators (NAA, IBA and MT) and the amounts were adjusted, the other conditions and the procedure being as in example 5. Specific types and amounts are shown in Table 8, and rooting test results are shown in tables 9 to 12 and FIG. 5.
Table 8 two-step rooting method orthogonal design test factors and level
TABLE 9 rooting Property under different treatments and multiple comparisons (two-step rooting method)
The injection is that A: days of dark treatment; b: NAA concentration; c: IBA concentration; d: MT concentration; different letters in the same column represent significant differences (p.ltoreq.0.05).
Table 10 analysis of variance of various indexes of rooting of Diospyros kaki (two-step rooting method)
Note that x represents a significant correlation with p < 0.05.
Table 11 various index membership function values of rooting of small fruit sweet persimmon after different treatments (two-step rooting method)
Table 12 comprehensive membership function range analysis (two-step rooting method)
Note that L m is the sum of different levels p values for the same factor. lm is the average of p values at different levels for the same factor, and R m is very bad.
Effect of dark treatment time and plant growth regulator concentration on rooting in the dip treatment:
Table 9 multiple comparative analysis and fig. 5 shows that root length and root surface area effects are best for treatment group 4: dark treatment is carried out for 10 days by 0.5mg/L NAA+0.2mg/L IBA+5mg/L MT, the root length reaches 24.14cm, and the root surface area reaches 9.74cm 2. The root system volume effect is preferably treatment group 3:1mg/L NAA+1.5mg/L IBA+5mg/L MT dark treatment is carried out for 5 days, and the root system volume reaches 0.29cm 3. The highest rooting rate treatment group 7 is that NAA+1.5mg/L IBA+0.1mg/L MT is subjected to dark treatment for 15 days, and the rooting rate reaches 80%.
The analysis of variance results in table 10 shows that the influences of different treatment factors on root length, root surface area, root volume and rooting rate of the tissue culture seedling are different, and the F ratio of the treatment factors of the root length is MT mass concentration, dark treatment days, NAA mass concentration and IBA mass concentration from large to small; the ratio of F of each treatment factor of the surface area and the volume of the root system is NAA mass concentration, dark treatment days, MT mass concentration and IBA mass concentration from large to small; the ratio of F of each treatment factor of the rooting rate is from big to small, and the ratio is the number of dark treatment days, NAA mass concentration, MT mass concentration and IBA mass concentration. NAA, IBA, MT mass concentration and dark treatment days obviously influence the length of a root system (p is less than 0.05), except IBA, the dark treatment days, NAA mass concentration and MT mass concentration obviously influence the surface area and the volume of the root system, and the rooting rate is obviously different from the dark treatment days and NAA mass concentration.
As can be seen from the analysis, the dark treatment days in the two-step rooting of the invention are preferably 5-15 days, the NAA concentration is preferably 0.5-1 mg/L (excluding the treatment group 1, the treatment group 5 and the treatment group 8), the IBA concentration is preferably 0.2-1.5 mg/L, and the MT concentration is preferably 0.1-5 mg/L; the rooting rate of the invention reaches 51.85-80.00%, the average total length of the most robust root system reaches 10.79-24.14 cm, the average surface area of the root system is 4.37-9.74 cm 2, and the average volume of the root system is 0.13-0.29 cm 3.
Comprehensive evaluation of rooting Effect
(1) By the analysis of table 6 in example 4, the comprehensive membership function value of each treatment of the dipping method was 2> treatment 3> treatment 1> treatment 6> treatment 5> treatment 4> treatment 7> treatment 9> treatment 8, and the comprehensive membership function value was at most 2. The minimum R value was determined based on the average p of each treatment, as shown in Table 7, and the maximum R value was 3.10 for the growth regulator species (0.56) for the treatment time (0.67), indicating that the growth regulator species had the greatest effect on rooting of the Diospyros kaki, the second for the growth regulator species, and the least effect on the treatment time, and the optimal regimen was A 1B3C2 (dipping treatment 2), i.e., 200mg/L NAA treatment for 10min.
(2) By the analysis of table 11 in example 6, the comprehensive membership function value of each treatment of the two-step rooting method was treated with 4> treatment 3> treatment 6> treatment 9> treatment 2> treatment 7> treatment 5> treatment 8> treatment 1, and the comprehensive membership function value was treated with 4 at the highest. The minimum R value was determined from the mean value p of each treatment, as shown in Table 12, and was NAA concentration (1.16) > MT concentration (0.96) > IBA concentration (0.86) > day of dark treatment (0.75), indicating that NAA concentration had the greatest effect on rooting of Diospyros kaki, MT concentration was secondary, IBA concentration was tertiary, day of dark treatment, least effect, and the preferred protocol was A 2B2C1D3, i.e., 0.5mg/L NAA+0.2mg/L IBA+5mg/L MT, was added to the minimal medium for 10 days of dark treatment.
(3) The combination of the dipping method proves that the independent MT treatment has no obvious rooting effect, even has far less rooting effect than the independent IBA treatment, but can generate synergistic effect when being compounded with NAA and IBA for two-step rooting, and effectively promote plant root to be strong on the premise of ensuring rooting rate, thereby being beneficial to improving survival rate.
Example 7 (examination of the influence of closed-flask seedling hardening time on the domestication of tissue-cultured seedlings obtained by the dipping method)
Example 7 differs from example 1, step 4) only in that: the closed flask seedling hardening time is adjusted, and other conditions and steps are the same as in example 1. The closed flask seedling hardening time and specific test results are shown in table 13 below.
TABLE 13 influence of closed-bottle seedling hardening on survival rate of domesticated and transplanted Diospyros kaki (dipping method)
Example 8 (investigating the influence of closed-flask seedling hardening time on transplanting and domestication of tissue culture seedlings obtained by two-step rooting method)
Example 8 differs from example 5, step 4) only in that: the closed flask seedling hardening time was adjusted, and other conditions and steps were the same as in example 5. The closed flask seedling time and specific test results are shown in table 14 below.
Table 14 influence of closed-bottle seedling hardening on survival rate of domesticated and transplanted Diospyros kaki (two-step rooting method)
In tissue culture of woody plants, the tissue culture seedlings take nutrient substances in a culture medium as nutrients for a long time, so that the seedlings mainly grow in a heterotrophic mode and photosynthesis is auxiliary. Therefore, in domestication, the tissue culture seedlings are firstly transferred from a heterotrophic state to a state mainly growing by photosynthesis. The tissue culture seedling is in a relatively closed state in a culture bottle, the relative humidity is high (80% -95%), the illumination is insufficient (the illumination of a general container is about 1500-3000lx and is far lower than the outdoor natural illumination), the tissue culture seedling is gradually transited from a high-humidity and weak-illumination state to a natural environment state during transplanting and domestication, the germ propagation speed is high in a high-temperature and high-humidity state, and the pollution rate of the tissue culture seedling is increased. As shown in the test results of Table 13, the seedlings of the small fruit sweet persimmon tissue culture obtained by the dipping method are subjected to closed-bottle seedling hardening for 7 days and then opened for 3 days, the survival rate of the seedlings is improved from 32% to 52.5% (3 d relative to closed-bottle seedling hardening), while the seedlings of the small fruit sweet persimmon tissue culture obtained by the two-step rooting method are subjected to closed-bottle seedling hardening for 7 days and then opened for 3 days, and the survival rate of the seedlings is improved from 36% to 57.33%; in addition, the seedling hardening off in a closed bottle with too long time can be accelerated under strong light, so that the seedling hardening off time is not suitable to be too long.
Compared with the prior art, the optimal concentration and the using time of the disinfectant play an important role in establishing a fast-growing system of persimmon tissue culture, and in the system, the research shows that the pollution degree is higher after the persimmon is treated by sodium hypochlorite with the concentration of 0.5 percent, the survival rate of the explant of the persimmon is lower, the pollution degree is reduced after the persimmon is treated by sodium hypochlorite with the concentration of 2 percent, but the explant is damaged during disinfection, the survival rate is correspondingly reduced, and the pollution rate and the disinfection mortality rate are controlled in a certain range when the persimmon is treated by sodium hypochlorite with the concentration of 1 percent for 6 minutes, so that the survival rate is highest. Unlike the conventional effect that betaine is often used as a protective agent against abiotic stress in plants, the present invention adds 100-1000. Mu. Mol/L betaine to the proliferation medium to increase the proliferation rate of the small fruit sweet persimmon, and particularly, 1000. Mu. Mol/L betaine is added to the medium, so that the proliferation effect of the small fruit sweet persimmon is optimal, which has not been reported before in persimmon. The invention aims at solving the problem of difficult rooting, two methods are utilized to explore the sweet persimmon with small fruits, one is to dip the root of young shoots with plant growth regulators, and the other is to perform dark culture on a culture medium containing the growth regulators at the initial stage of rooting and then transfer the culture medium without the growth regulators to root, although the dipping method has the advantage of saving the use of the culture medium and the rooting rate reaches 90% at most compared with the two-step rooting method, the pollution rate is increased due to long-time exposure, and the two-step rooting method is used for replacing the culture medium for one time, but the pollution rate is reduced and the rooting rate reaches 80% at most, so the invention is preferably a two-step rooting method.
Research shows that the growth and development of plant root system is strictly regulated by plant hormone, and the growth hormone, cytokinin, ethylene (ETH) and other processes regulate and control the side root generation, root cell elongation, root hair formation and the like. Many researches show that the adventitious persimmon root belongs to an induction type root primordium, the root primordium can be induced by a certain stimulus, the exogenous auxin is used as a regulating substance for inducing rooting, the level of plant endogenous hormone can be changed, the balance of the endogenous hormone is regulated so as to play a role in the growth and development of root system cells, the IBA, NAA and melatonin are adopted to cooperate to induce the rooting of persimmon tissue culture seedlings, an important role is played in the rooting of persimmon tissue culture seedlings, a positive synergistic effect is generated, and an orthogonal experimental design is utilized, and the optimal conditions of dipping treatment and two-step rooting treatment are explored by adopting NAA, IBA and MT as growth regulators in a dipping treatment optimal scheme of A 1B3C2, namely 200mg/L NAA treatment for 10min; the best scheme of the two-step rooting method is A 2B2C1D3, namely 0.5mg/L NAA+0.2mg/L IBA+5mg/L MT is added to the minimal medium for dark treatment for 10 days. Finally, the seedling hardening days of bottle closing and cover opening are controlled, and the tissue culture seedlings of the small-fruit sweet persimmon are successfully domesticated, so that a complete persimmon in-vitro culture propagation system is constructed.
As shown in FIG. 6, the complete persimmon in-vitro culture propagation system constructed by the invention comprises the following steps:
(1) The primary culture includes: a. dormancy buds; b, sterilizing with 75% alcohol; c, sterilizing with 1% sodium hypochlorite; d. the surgical knife strips off a plurality of layers of scales outside the bud; e. inoculating into a culture medium; f. adventitious bud formation and elongation (30 d culture); g. primary culturing stem segments (1-1.5 cm) with 4-5 leaves; h. primary culturing stem segments with buds; i. performing primary culture for 30d, and then performing tissue culture seedling;
(2) Proliferation culture includes: j. subculturing for 30 days and then carrying out bud stem segment; k. the stem with 4-5 leaves (2-3 cm long) after 30d of subculture;
(3) Rooting culture: i, rooting tissue culture seedlings;
(4) Domestication and transplanting: m, domesticating tissue culture seedlings; n. transplanting the plants after 2 months.
The invention solves the problems that the variety of the main cultivated persimmon-rich line is not compatible with the grafting of the monarch and the common stock, and greatly restricts the popularization of the persimmon and the healthy development of the persimmon industry. Experiments are carried out by utilizing the combined hormone of NAA, IBA and melatonin, and the synergistic effect of the three hormones is found to obviously improve the rooting rate of the tissue culture seedlings of the Diospyros kaki and to strengthen the root system; at present, the period required by the actual propagation (seed propagation) seedling culture is longer, the consistency of the obtained seedlings is poor, the time required by the seedlings to reach a grafting state is more than one year, the seedlings can be used as stock for grafting after being transplanted to a field for 6-8 months (the thickness can reach about 4mm after 6 months), and therefore, the whole period of subculture, rooting, transplanting and domestication is about 11 months, and the seedling culture period is greatly shortened.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.
Claims (7)
1. The tissue culture and rapid propagation method of the small-fruit sweet persimmon is characterized by comprising the following steps of:
(1) Primary culture: taking the stem with buds, sterilizing, stripping a plurality of layers of scales outside the full dormant buds, inoculating the stem with buds into a primary culture medium, and culturing to obtain primary tissue culture seedlings; the primary culture medium consists of MS culture medium with halved nitrogen element, ZT of +1 mg/L, IAA of +0.1 mg/L, sucrose of +30 mg/L, agar of +7 g/L and PVP-40 of 0.6 g/L;
(2) Proliferation culture: cutting the primary tissue culture seedling into stem segments, inoculating the stem segments into a proliferation culture medium containing betaine for culture to obtain a root-free tissue culture seedling; the proliferation culture medium containing betaine consists of DKW+1 mg/L ZT+0.1 mg/L IAA+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40+100-1000 [ mu ] mol/L betaine;
(3) Rooting culture: rooting culture is carried out on the rooting-free tissue culture seedlings by adopting a dipping rooting method or a two-step rooting method, so that rooting tissue culture seedlings are obtained; wherein, the first plant growth regulator adopted in the dipping rooting method is NAA or IBA; the second plant growth regulator adopted in the two-step rooting method is a mixture of NAA, IBA and MT;
The dipping rooting method is to firstly soak the basal part of the rooting-free tissue culture seedling with a first plant growth regulator, then carry out dark treatment for 5-15 d, and then transfer the basal part into a basic culture medium A for rooting culture for 30-50 d; when the first plant growth regulator is NAA, the concentration is 50-200 mg/L, and the soaking time is 5-15 min; when the first plant growth regulator is IBA, the concentration is 100-200 mg/L, and the soaking time is 5-10 min; the basic culture medium A consists of 1/2 MS+1 mg/L active carbon+30 mg/L sucrose+7 g/L agar+0.6 g/L PVP-40;
The two-step rooting method is that firstly, the radicle tissue culture seedling is inoculated into a basic culture medium B, after being subjected to dark treatment for 5-15 d, the radicle tissue culture seedling is transferred into the basic culture medium A for rooting culture for 30-50 d; the basic culture medium B consists of basic culture medium A+0.5-1 mg/L NAA+0.2-1.5 mg/L IBA+0.1-5 mg/L MT;
(4) Domestication and transplanting: taking rooting tissue culture seedlings, closing bottles, hardening the seedlings, opening the cover, hardening the seedlings, transplanting the seedlings to seedling trays for culturing until the seedlings survive, and transplanting the seedlings to a field.
2. The tissue culture rapid propagation method of small fruit sweet persimmon according to claim 1, wherein in the step (1), the bud-bearing stem section is obtained by collecting strong and bud-filled branches in winter; the sterilization is to dip and wash the bud stem with Tween-20 for 1-2 min and then clean the bud stem with sterile water; the disinfection is to soak the sterilized bud-bearing stem in 75% alcohol solution for 25-35 s, disinfect in 0.5-1.2% sodium hypochlorite solution for 5-7 min and then wash.
3. The method for tissue culture and rapid propagation of small fruit sweet persimmon according to claim 1, wherein in the step (1), 2-3 layers of scales outside full and dormant buds are stripped and inoculated into a primary culture medium; the culture conditions of the primary culture are that the primary culture is carried out in a tissue culture room with the temperature of 25+/-2 ℃ and the illumination intensity of 1000-3000 lx and the illumination time of 16 h/d, and the culture time is 30-60 d.
4. The method for tissue culture and rapid propagation of Diospyros kaki according to claim 1, wherein in the step (2), the primary tissue culture seedling is cut into stem segments of 1-1.5 cm.
5. The method for tissue culture and rapid propagation of Diospyros kaki according to claim 1, wherein in the step (2), the culture conditions for the propagation culture are the same as those for the primary culture, and the culture time is 30 to 60 days.
6. The tissue culture rapid propagation method of small fruit sweet persimmon according to claim 1, wherein in the step (4), the closed bottle seedling hardening time is 3-10 d, and the open cover seedling hardening time is 2-4 d.
7. The method for tissue culture and rapid propagation of small fruit sweet persimmon according to claim 1, wherein in the step (4), the culture medium transplanted to the seedling tray adopts mixed soil of seedling medium and vermiculite, and the 1000 times carbendazim is used for soil mixing before cultivation; after transplanting to seedling tray, covering with air-permeable heat-insulating cover, adding water into tray; every 7 d times of carbendazim solution is sprayed, and the mixture is placed under the natural illumination condition of 23-25 ℃ for culture until survival.
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| CN115152509A (en) * | 2022-07-13 | 2022-10-11 | 山东省烟台市农业科学研究院 | Method for promoting persimmon rootstock seed germination and seedling growth |
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| CN115152509A (en) * | 2022-07-13 | 2022-10-11 | 山东省烟台市农业科学研究院 | Method for promoting persimmon rootstock seed germination and seedling growth |
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